This invention relates to sample preparation devices having small dimensions for facilitating the efficient preparation of microvolume test samples, e.g., of whole blood, for the determination and/or processing of analytes present therein. The present invention also relates to test systems including such devices, together with devices of similar dimensions which are designed, for example, to perform various assay protocols as well as analyses involving amplification of pre-selected polynucleotides, such as polymerase chain reaction (PCR).
In recent decades the art has developed a large number of protocols, test kits, and devices for conducting analyses on biological samples for various diagnostic and monitoring purposes. Immunoassays, immunometric assays, agglutination assays, analyses involving polynucleotide amplification reactions, various ligand-receptor interactions, and differential migration of species in a complex sample all have been used to determine the presence or quantity of various biological molecules or contaminants, or the presence of particular cell types.
Recently, small, disposable devices have been developed for handling biological samples and for conducting certain clinical tests. Shoji et al. reported the use of a miniature blood gas analyzer fabricated on a silicon wafer. Shoji et al., Sensors and Actuators, 15: 101-107 (1988). Sato et al. reported a cell fusion technique using micromechanical silicon devices. Sato et al., Sensors and Actuators, A21-A23: 948-953 (1990). Ciba Corning Diagnostics Corp. (USA) has manufactured a microprocessor-controlled laser photometer for detecting blood clotting.
Micromachining technology originated in the microelectronics industry. Angell et al., Scientific American, 248: 44-55 (1983). Micromachining technology has enabled the manufacture of microengineered devices having structural elements with minute dimensions, ranging from tens of microns (the dimensions of biological cells) to nanometers (the dimensions of some biological macromolecules). Most experiments reported to date involving such small structures have involved studies of micromechanics, i.e., mechanical motion and flow properties. The potential capability of such devices has not been exploited fully in the life sciences.
Brunette (Exper. Cell Res., 167: 203-217 (1986) and 164: 11-26 (1986)) studied the behavior of fibroblasts and epithelial cells in grooves in silicon, titanium-coated polymers and the like. McCartney et al. (Cancer Res., 41: 3046-3051 (1981)) examined the behavior of tumor cells in grooved plastic substrates. LaCelle (Blood Cells, 12: 179-189 (1986)) studied leukocyte and erythrocyte flow in microcapillaries to gain insight into micro-circulation. Hung and Weissman reported a study of fluid dynamics in micromachined channels, but did not produce data associated with an analytical device. Hung et al., Med. and Biol. Engineering, 9: 237-245 (1971); and Weissman et al., Am. Inst. Chem. Eng. J., 17: 25-30 (1971). Columbus et al. utilized a sandwich composed of two orthogonally orientated v-grooved embossed sheets in the control of capillary flow of biological fluids to discrete ion-selective electrodes in an experimental multi-channel test device. Columbus et al., Clin. Chem., 33: 1531-1537 (1987). Masuda et al. and Washizu et al. have reported the use of a fluid flow chamber for the manipulation of cells (e.g., cell fusion). Masuda et al., Proceedings IEEE/IAS Meeting, pp. 1549-1553 (1987); and Washizu et al., Proceedings IEEE/IAS Meeting, pp. 1735-1740 (1988). The art has not fully explored the potential of using microengineered devices for the determination of analytes in fluid samples, particularly in the area of biological analyses.
Biological analyses utilizing polynucleotide amplification techniques are well known (See e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, pp. 14.1-14.35). One such technique is PCR amplification, which can be performed on a DNA template using a thermostable DNA polymerase, e.g., Taq DNA polymerase (Chien et al., J. Bacteriol., 127: 1550 (1976)), nucleoside triphosphates, and two oligonucleotides with different sequences, complementary to sequences that lie on opposite strands of the template DNA and which flank the segment of DNA that is to be amplified ("primers"). The reaction components are cycled between a higher temperature (e.g., 94.degree. C.) for dehybridizing double stranded template DNA, followed by lower temperatures (e.g., 65.degree. C.) for annealing and polymerization. A repeated reaction cycle between dehybridization, annealing and polymerization temperatures provides approximately exponential amplification of the template DNA. Machines for performing automated PCR chain reactions using a thermal cycler are available (Perkin Elmer Corp.)
PCR amplification has been applied to the diagnosis of genetic disorders (Engelke et al., Proc. Natl. Acad. Sci., 85: 544 (1988), the detection of nucleic acid sequences of pathogenic organisms in clinical samples (Ou et al., Science, 239: 295 (1988)), the genetic identification of forensic samples, e.g., sperm (Li et al., Nature, 335: 414 (1988)), the analysis of mutations in activated oncogenes (Farr et al., Proc. Natl. Acad. Sci., 85: 1629 (1988)) and in many aspects of molecular cloning (Oste, BioTechniques, 6: 162 (1988)). PCR assays can be used in a wide range of applications such as the generation of specific sequences of cloned double-stranded DNA for use as probes, the generation of probes specific for uncloned genes by selective amplification of particular segments of cDNA, the generation of libraries of cDNA from small amounts of mRNA, the generation of large amounts of DNA for sequencing, and the analysis of mutations. There is a need for convenient, rapid systems for performing polynucleotide amplification, which may be used clinically in a wide range of potential applications in clinical tests such as tests for paternity, and for genetic and infectious diseases.
Current analytical techniques utilized for the determination of microorganisms are rarely automated, usually require incubation in a suitable medium to increase the number of organisms, and generally employ visual and/or chemical methods to identify the strain or sub-species of interest. The inherent delay in such methods frequently necessitates medical intervention prior to definitive identification of the nature of an infection. In industrial, public health or clinical environments, such delays may have unfortunate consequences. There is a need for convenient systems for the rapid detection of microorganisms.
It is an object of the present invention to provide sample preparation devices for use with related analytical devices which enable rapid and efficient analysis of sample fluids, based on very small volumes, and determination of substances present therein at very low concentrations. Another object is to provide easily mass produced, disposable, small (e.g., less than 1 cc in volume) devices having microfabricated structural elements capable of facilitating rapid, automated analyses of preselected molecular or cellular analytes, including intra-cellular molecules, such as DNA, in a range of biological and other applications. It is a further object of the invention to provide a variety of such devices that individually can be used to implement a range of rapid clinical tests, e.g., tests for viral or bacterial infection, genetic screening, sperm motility, blood parameters, contaminants in food, water, or body fluids, and the like.